The Hippo pathway drives the cellular response to hydrostatic pressure

Abstract Cells need to rapidly and precisely react to multiple mechanical and chemical stimuli in order to ensure precise context‐dependent responses. This requires dynamic cellular signalling events that ensure homeostasis and plasticity when needed. A less well‐understood process is cellular response to elevated interstitial fluid pressure, where the cell senses and responds to changes in extracellular hydrostatic pressure. Here, using quantitative label‐free digital holographic imaging, combined with genome editing, biochemical assays and confocal imaging, we analyse the temporal cellular response to hydrostatic pressure. Upon elevated cyclic hydrostatic pressure, the cell responds by rapid, dramatic and reversible changes in cellular volume. We show that YAP and TAZ, the co‐transcriptional regulators of the Hippo signalling pathway, control cell volume and that cells without YAP and TAZ have lower plasma membrane tension. We present direct evidence that YAP/TAZ drive the cellular response to hydrostatic pressure, a process that is at least partly mediated via clathrin‐dependent endocytosis. Additionally, upon elevated oscillating hydrostatic pressure, YAP/TAZ are activated and induce TEAD‐mediated transcription and expression of cellular components involved in dynamic regulation of cell volume and extracellular matrix. This cellular response confers a feedback loop that allows the cell to robustly respond to changes in interstitial fluid pressure.

. Cytoskeletal implications in the cellular response to hydrostatic pressure.
A Average change in cell volume in response to 100 mbar cyclic hydrostatic pressure in WT and LATS1/2 DKO clones. Each dot represents a single cell. Data pooled from three independent experiments. Error bars represent mean AE 95% CI. Kruskal-Wallis test with Dunn's post-hoc. P > 0.9999 (for all comparisons). B Average change in cell volume in response to 100 mbar cyclic hydrostatic pressure in WT and MST1/2 DKO. Each dot represents a single cell. Data pooled from three independent experiments. Error bars represent mean AE 95% CI. Mann-Whitney U test. P = 0.0781. C HEK293A NF2 KO steady-state volume compared to WT cells. Each dot represents a single cell and Error bars represent mean AE 95% CI. Data pooled from three independent experiments. Mann-Whitney U test. ***P < 0.001. D PhosTag-based Western blot probing for YAP reveals dephosphorylation of YAP in response to 0.5 lM latrunculin B (LB) and 2 lM cytochalasin D (CD) treatment in WT cells. E Comparison of average change in cell volume measured by DHM in response to 100 mbar cyclic hydrostatic pressure with 2 lM latrunculin B treatment in WT, Y/T DKO and LATS1/2 DKO cells. Each cell represents a single cell and error bars represent mean AE 95% CI. Data pooled from four independent experiments. Mann-Whitney U test. ***P < 0.001 (WT), ***P < 0.001 (Y/T DKO) and *P = 0.0331 (LATS1/2 DKO).  A Lysates from cells treated for 40 min with Torin (1 lM) or Rapamycin (0.5 lM) compared to control cells were analysed by immunoblots for the levels of the mTORC1 substrate S6K, pS6K and GAPDH (loading control). Note the levels of pS6K are drastically decreased in Torin-and Rapamycin-treated HEK293A cells, highlighting that these drugs effectively inhibits S6K phosphorylation. B Cells treated with Torin (1 lM) and Rapamycin (0.5 lM) as in (A) and analysed by DHM to obtain their optical cellular volume. Each dot represents a single cell from three independent experiments. Forty minutes Torin or Rapamycin treatment does not affect steady-state cell volume. Error bars represent mean AE 95% CI. Kruskal-Wallis test with Dunn's post-hoc. P = 0.5709 (con vs. Torin), P > 0.9999 (con vs. Rapamycin) and P > 0.9999 (Torin vs. Rapamycin). C WT cells treated with Torin (1 lM) and Rapamycin (0.5 lM) as in (A) and imaged using DHM while being subjected to cyclic 0.1 Hz 100 mbar fluid pressure. Forty minutes Torin (1 lM) and Rapamycin (0.5 lM) treatment has no effect on cell volume changes in response to hydrostatic pressure. Each dot represents a single cell from three independent experiments. Error bars represent mean AE 95% CI. Kruskal-Wallis test with Dunn's post-hoc. P = 0.5321 (con vs. Torin), P > 0.9999 (con vs. Rapamycin) and P = 0.4860 (Torin vs. Rapamycin). D Western blot confirming no CAV1 protein expression in CAV1 KO HEK293A cells. E CAV1 KO cellular response to cyclic 0.1 Hz 200 mbar hydrostatic pressure compared to WT. Each dot represents a single cell from three independent experiments and error bars represent mean AE 95% CI. Mann-Whitney U test. P = 0.4904. F Relative CAV1 expression levels of CAV1 knockdown clones #1 and #2 from four independent experiments. Mann-Whitney U test. Error bars represent mean AE SD. **P = 0.0079. G Western blot confirming reduction in total CAV1 protein levels in shCAV1 clones #1 and #2. H shRNA CAV1 knockdown cell response to cyclic 0.1 Hz 100 mbar hydrostatic pressure compared to WT. Each dot represents a single cell and error bars represent mean AE 95% CI. Data pooled from four independent experiments. Kruskal-Wallis test with Dunn's post-hoc. P > 0.9999 (vector vs. shCAV#1), P = 0.1892 (vector shCAV#2) and P = 0.0812 (shCAV#1 vs. shCAV#2).
Source data are available online for this figure.
The  Figure EV4. Endocytosis rates upon oscillating hydrostatic pressure.
A Confocal image of fluorescently labelled transferrin (red) uptake by 143B cells under steady-state conditions and in response to cyclic 0.1 Hz 200 mbar hydrostatic pressure. Cells labelled for Hoechst (blue). Scale bar = 20 lm. B Quantification of transferrin uptake in 143B cells treated with hydrostatic pressure compared to steady state from images as in A. Each dot represents a single cell and error bars represent mean AE 95% CI. Data from four independent experiments. Mann-Whitney U test. *** P < 0.001. C Confocal IF images of HEK293A cells. Cells are labelled with Dextran (green) and Hoechst (blue). HEK293A WT dextran uptake in response to 10 min (left) and 30 min (right) cyclic hydrostatic pressure compared to steady state (Con). Scale bar = 20 lm. D Confocal images of Y/T DKO HEK293A cells. Cells are labelled with Dextran (green) and Hoechst (blue). Y/T DKO HEK293A dextran uptake in response to 10 min (left) and 30 min (right) cyclic hydrostatic pressure compared to steady state (Con). Scale bar = 20 lm. E Confocal images of LATS1/2 DKO HEK293A cells. Cells are labelled with Dextran (green) and Hoechst (blue). LATS1/2 DKO HEK293A dextran uptake in response to 10 min (left) and 30 min (right) cyclic hydrostatic pressure compared to steady state (Con). Scale bar = 20 lm. Relative dextran uptake ns Dextran uptake (30min) and H.P.
Source data are available online for this figure. The